Recurring BALB/c mouse lung inflammatory responses to episodic allergen exposure.

This study detailed the sequence of recurring inflammatory events associated with episodic allergen exposures of mice resulting in airway hyperreactivity, sustained inflammation, goblet-cell hyperplasia, and fibrogenesis that characterize a lung with chronic asthma. Ovalbumin (OVA)-sensitized female BALB/c mice were exposed to saline-control or OVA aerosols for 1 h per day for episodes of 3 d/wk for up to 8 wk. Lung inflammation was assessed by inflammatory cell recoveries using bronchoalveolar lavages (BAL) and tissue collagenase dispersions. Cell accumulations were observed within airway submucosal and associated perivascular spaces using immunohistochemical and tinctorial staining methods. Airway responsiveness to methacholine aerosols were elevated after 2 wk and further enhanced to a sustained level after wk 4 and 8. Although by wk 8 diminished OVA-induced accumulations of eosinophils, neutrophils, and monocyte-macrophages were observed, suggesting diminished responsiveness, the BAL recovery of lymphocytes remained elevated. Airway but not perivascular lesions persisted with a proliferating cell population, epithelial goblet-cell hyperplasia, and evidence of enhanced collagen deposition. Examination of lung inflammatory cell content before the onset of the first, second, and fourth OVA exposure episodes demonstrated enhancements in residual BAL lymphocyte and BAL and tissue eosinophil recoveries with each exposure episode. Although tissue monocyte-macrophage numbers returned to baseline prior to each exposure episode, the greatest level of accumulation was observed after wk 4. These results provide the basis for establishing the inflammatory and exposure criteria by which episodic environmental exposures to allergen might result in the development of a remodeled lung in asthma.

Hyperoxic sheep pulmonary microvascular endothelial cells generate free radicals via mitochondrial electron transport.

Free radical generation by hyperoxic endothelial cells was studied using electron paramagnetic resonance (EPR) spectroscopy and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the radical species produced, whether mitochondrial electron transport was involved, and the effect of the radical generation on cell mortality. Sheep pulmonary microvascular endothelial cell suspensions exposed to 100% O2 for 30 min exhibited prominent DMPO-OH and, occasionally, additional smaller DMPO-R signals thought to arise from the trapping of superoxide anion (O2-.), hydroxyl (.OH), and alkyl (.R) radicals. Superoxide dismutase (SOD) quenched both signals suggesting that the observed radicals were derived from O2-.. Studies with deferoxamine suggested that the generation of .R occurred secondary to the formation of .OH from O2-. via an iron-mediated Fenton reaction. Blocking mitochondrial electron transport with rotenone (20 microM) markedly decreased radical generation. Cell mortality increased slightly in oxygen-exposed cells. This increase was not significantly altered by SOD or deferoxamine, nor was it different from the mortality observed in air-exposed cells. These results suggest that endothelial cells exposed to hyperoxia for 30 min produce free radicals via mitochondrial electron transport, but under the conditions of these experiments, this radical generation did not appear cause cell death.

Triacylglycerol hyrolysis in the isolated, perfused rat lung.

We have studied the mechanism of hydrolysis of labeled long-chain triacylglycerols in the isolated, ventilated, perfused rat lung. Hydrolysis of emulsified tri[3H]oleate or doubly labeled [3H]glyceryl, tri[14C]oleate was measured by quantitation of [3H]oleate or of [14C]oleate and [3H]glycerol released into the perfusate. Hydrolysis was directly proportional to the initial triacylglycerol concentration of the perfusate in the range of 0.30 to 2.2 mM. The release of free fatty acids was linear after an initial lag period, the length of which was inversely proportional to the triacylglycerol concentration. Studies with doubly labeled [3H]glyceryl, tri[14C]oleate showed that, during triacylglycerol hydrolysis, the molar ratio of free fatty acid to glycerol released is close to 1, suggesting that about one-third of the fatty acids hydrolyzed is released into the pulmonary circulation. The earlier appearance of free fatty acid than glycerol in the venous effluent indicates that the first step in triacylglycerol hydrolysis occurs at the endothelial surface. In order to investigate the role of lipoprotein lipase in this process, we administered heparin, which leads to immediate release of lipoprotein lipase from the endothelium to the circulation in vivo, 10 min and 4 h before isolation and perfusion of the lungs. Heparin administration 10 min prior to perfusion led to marked release of lipoprotein lipase from the lungs and completely abolished the subsequent hydrolysis of circulating triacylglycerols. Perfusions carried out 4 h after heparin administration show that in the lung, endothelial lipoprotein lipase levels did not return to normal within 4 h after heparin administration. The data show that circulating triacylglycerols are hydrolyzed by endothelial lipoprotein lipase during passage through the lung.

Pyruvate metabolism of perfused rat lungs after exposure to 100% oxygen.

Previous studies with lung homogenates have suggested that pulmonary O2 toxicity is in part a result of inhibited mitochondrial energy metabolism. In this study, mitochondrial metabolism was determined by measurements of 14CO2 production from [1-14C]-pyruvate in perfused lungs, isolated after 0, 3, 6, 12, and 24 h of exposure to 100% O2. Measurements were made under normal and stimulated conditions brought about by uncoupling oxidative phosphorylation with 2,4-dinitrophenol (DNP). Lungs were ventilated with 5% CO2 in O2 and perfused for 100 min with 12.5 mM 14C labeled pyruvate. Unexposed lungs gave a linear rate of 14CO2 production of 121 +/- 16 mumol/h/g dry wt (n = 5), which was maximally stimulated 84% by perfusion with 0.8 mMDNP. Twenty-four hours of exposure to 100% O2 did not significantly affect 14CO2 production. In contrast, DNP failed to significantly stimulate pyruvate metabolism to CO2 in lungs exposed for greater than 3 h to 100% O2. These latter data suggested that O2 exposure makes lung mitochondria unable to respond to increased ATP demands associated with DNP uncoupling. Compromised energy metabolism is therefore an important early event in O2 toxicity.

Rat lung glucose metabolism after 24 h of exposure to 100% oxygen.

Previous studies with lung homogenates and isolated cells have suggested oxygen cell injury results from the inhibition of key enzymes involved in both cytosolic and mitochondrial energy generation. In this study, the extent and pattern of metabolism of D-[U-14C, 5-3H]glucose was examined in perfused lungs isolated from rats before and after 24 h of in vivo exposure to 100% O2. Lung ATP levels after O2 exposure were maintained by a 53% increase in glucose utilization from an unexposed control value of 18.0 +/- 3.2 to 27.5 +/- 3.0 mumol 3H2O.h-1.g dry wt-1, accounted for by an enhanced rate of lactate plus pyruvate production from 15.7 +/- 2.0 to 32.7 +/- 4.1 mumol.h-1.g dry wt-1 with no alteration in lactate-to-pyruvate ratio. CO2 production was unaltered from a control rate of 27.5 +/- 4.0 14CO2 mumol.h-1.g dry wt-1. Maximal rates of glucose metabolism were determined by perfusion with 0.8 mM dinitrophenol, giving for air-exposed lungs a rate of 53.5 +/- 5.0 mumol 3H2O.h-1.g dry wt-1 and increased lactate plus pyruvate and 14CO2 production rates of 46.5 +/- 6.5 and 128.3 +/- 19.6 mumol.h-1.g dry wt-1, respectively. Although this maximal rate of glucose utilization was unaltered in oxygen-exposed lungs, lactate plus pyruvate production was further increased to 80.0 +/- 9.1 mumol.h-1.g dry wt-1 with a concomitant decrease in the dinitrophenol-induced rate of 14CO2 production to 81.5 +/- 9.2 mumol.h-1.g dry wt-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Ozone-induced airway hyperresponsiveness and loss of neuronal M2 muscarinic receptor function.

The effect of acute ozone exposure on the function of efferent parasympathetic nerves, M3 muscarinic receptors on airway smooth muscle, and inhibitory M2 muscarinic receptors on the parasympathetic nerves was studied. Immediately after exposure to 2.0 ppm ozone for 4 h, guinea pigs became hyperresponsive to electrical stimulation of the vagus nerves. The normal airway response to intravenous cholinergic agonists at this time demonstrates normal M3 receptor function. M2 muscarinic receptors on the nerves, which normally inhibit release of acetylcholine, were dysfunctional after ozone exposure, as demonstrated by the failure of the muscarinic agonist pilocarpine to inhibit, and the failure of the M2 antagonist gallamine to potentiate, vagally mediated bronchoconstriction. Thus, loss of inhibitory M2 muscarinic receptor function after ozone exposure potentiates release of acetylcholine from the vagus nerves, increasing vagally mediated bronchoconstriction. By 14 days, postozone responses to vagal nerve stimulation were not different from those of air-exposed animals and the function of the neuronal M2 muscarinic receptor was normal, confirming that ozone-induced hyperresponsiveness is reversible.

The influence of polymorphonuclear leukocytes on altered pulmonary epithelial permeability during ozone exposure.

Ozone (O3), a pulmonary irritant, and a major toxic component of photochemical smog, is capable of inducing pulmonary inflammation characterized by recruitment of polymorphonuclear leukocytes (PMNs) into the lung. The recruited PMNs, in turn, can release toxic mediators and produce lung injury. The mechanism of ozone-induced changes in lung permeability remains unknown. It is our hypothesis that PMNs migrating into the lung play a significant role in the pathophysiology following O3 exposure and that increasing the number of PMNs coming into the lung will exaggerate the changes in lung permeability. To test this hypothesis, we induced an influx of PMNs into the lungs of Sprague-Dawley rats by intratracheal instillation of 1% rabbit serum and then exposed the animals to either 0.8 ppm O3 or filtered air for 3 h. Control animals were intratracheally instilled with phosphate-buffered saline (PBS) and simultaneously exposed to O3 or filtered air in the same manner as the serum-treated animals. The animals were sacrificed and the lungs lavaged 10-12 h after exposure. The bronchoalveolar lavage fluid (BALF) was analyzed for albumin and protein, as indicators of permeability. In addition, BALF from the various groups was tested for its ability to alter epithelial resistance of pulmonary type II cells in culture. O3 exposure resulted in a significant increase in albumin and protein levels in the BALF as compared to air-exposed controls. The instillation of serum resulted in a significant increase in airway PMNs, but no significant elevations in albumin levels in both the O3 and air-exposed groups, as compared to PBS instillation. In vitro studies did not reveal a differential BALF effect on epithelial resistance. The data demonstrate that an excessive neutrophilia in the lung is not matched by a comparable amplification of epithelial injury. It is therefore suggested that a simple elevation in PMN number in the air spaces, as that induced by serum instillation, does not necessarily augment the lung pathophysiology, but that a more complex interaction with O3 may be required for cellular activation and release of toxic products.

Lung tissue neutrophil content as a determinant of ozone-induced injury.

Short-term exposure of rats to ozone results in lung inflammation characterized by increased permeability damage and the infiltration of neutrophils into the airways. The present study compared these ozone-induced inflammatory responses in different strains of male rat, Brown Norway rats from Charles River Laboratories, Inc. (BN-CRL), and Harlan Sprague Dawley, Inc. (BN-HSD), and Fischer 344 (F344), Sprague-Dawley (SPD), and Wistar (WSTR) male rats from Hilltop Lab Animals, Inc. Ozone-induced permeability damage was indicated by recoveries of bronchoalveolar lavage fluid (BALF) albumin 20 h following single exposures of 6 h to either air or 1 ppm or 2 ppm O3. Although BALF albumin recoveries from air-exposed rats were not significantly different between strains, ozone exposures resulted in a range of enhancements of BALF albumin of 2-, 9-, 17-, 7-, and 20-fold following exposures of BN-CRL, BN-HSD, F344, SPD, and WSTR rats to 2 ppm ozone, respectively. Concomitant strain differences in the number of ozone-induced BAL-recoverable neutrophils were not observed, except for BN-CRL rats, which demonstrated significantly lower numbers. However, the degree of ozone-induced permeability damage did directly correspond to differences observed in the numbers of neutrophils and eosinophils in the peripheral blood and collagenase tissue digest of lavaged and perfused lungs prior to ozone exposure. Ozone-resistant BN-CRL rats exhibited the highest numbers of blood and lung tissue neutrophils and eosinophils when compared with ozone-susceptible WSTR rats exhibiting the lowest number of these granulocytes. These data suggested that the presence of high numbers of blood and tissue granulocytes at the onset of short-term ozone exposures might provide a certain degree of protection against subsequent pathological events.

In vivo efficacy of dendrimer-methylprednisolone conjugate formulation for the treatment of lung inflammation.

Dendrimers are an emerging class of nanoscale intracellular drug delivery vehicles. Methylprednisolone (MP) is an important corticosteroid used in the treatment (through inhalation) of lung inflammation associated with asthma. The ability of MP-polyamidoamine (PAMAM) dendrimer conjugate to improve the airway delivery was evaluated in a pulmonary inflammatory murine model that was based on an 11-fold enhancement of eosinophil lung accumulation following five daily inhalation exposures of sensitized mice to the experimental allergen, ovalbumin. MP was successfully conjugated to PAMAM-G4-OH dendrimer yielding 12 MP molecules per dendrimer, and further solubilized in lysine carrier. Five daily trans-nasal treatments with the carrier alone, free MP, and MP-dendrimer at 5 mg kg(-1) (on a drug basis) did not induce additional lung inflammation, although free MP decreased baseline phagocytic cell recoveries by airway lavage and tissue collagenase dispersion. MP treatments alone decreased ovalbumin-associated airway and tissue eosinophil recoveries by 71 and 47%, respectively. Equivalent daily MP dosing with MP-dendrimer conjugate further diminished these values, with decreases of 87% and 67%, respectively. These findings demonstrate that conjugation of MP with a dendrimer enhances the ability of MP to decrease allergen-induced inflammation, perhaps by improving drug residence time in the lung. This is supported by the fact that only 24% of a single dose of dendrimer delivered to the peripheral lung is lost over a 3-day period. Therefore, conjugation of drugs to a dendrimer may provide an improved method for retaining drugs within the lung when treating such inflammatory disorders as asthma.

Effect of 2 ppm ozone exposure on rat lung lipid fatty acids.

Based on in vitro studies, the initial damage to lung cells by ozone exposure is believed to result in part from the breakdown of lipid polyunsaturated fatty acids to aldehydes, ozonides, and peroxides. The present study measured lipid breakdown products in lungs isolated from rats pretreated with [1-14C]acetate 12 h before exposure for 4 h to either air or 2 ppm ozone. Lipid fatty acid breakdown was indicated by a 112% increase in thiobarbituric acid-reactive substances on ozone exposure and by changes in chemical and radioactive measurements of mono- and dicarboxylic acids formed by treatment of lipid fractions with hydrogen peroxide. Ozone exposure resulted in a 63% increase in recovery of short-chain fatty acids accounted for by increased recoveries of malonic acid by 37%, hexanoic acid by 47%, nonanoic acid by 118%, and azelaic acid by 107%. Recovery of glutaric acid was enhanced 15-fold by ozone exposure. Although decreases in tissue arachidonic acid could not be detected, oleic acid was significantly decreased by 36%. Recovery of radiolabel as short-chain fatty acids was increased by 65% on ozone exposure and was mainly accounted for by enhanced labeling of nonanoic and glutaric acid fractions. The failure to observe significant increases in 14C recovery in the other fractions suggested ozone-induced breakdown of unlabeled fatty acids. These results demonstrate the cleavage of unsaturated fatty acid double bonds following in vivo exposure of lungs to ozone. Breakdown of arachidonic and oleic acids was specifically identified by increased recoveries of glutaric and nonanoic acids, respectively.

Pulmonary cadmium oxide toxicity in the rat.

Although occupational exposures to cadmium have usually involved inhalation of insoluble cadmium oxide (CdO) particles, experimental studies of pulmonary cadmium toxicity have relied on aerosol exposures to soluble cadmium chloride particles. The present study describes a model of acute lung injury based on single 3-h exposures of rats to 0.5 and 5.3 mg/m3 CdO. Biochemical changes were correlated with pathological observations for 15 d postexposure to CdO. Four days following CdO exposure, histopathological observations included focal areas of epithelial hyperplasia, a mononuclear interstitial infiltrate, and increased numbers of alveolar macrophages. In the high-dose group, these changes were correlated with increases in tissue protein and DNA contents of 217% and 195% of controls, respectively. While lungs from the low-dose exposures had returned to a normal appearance by 15 d postexposure, high-dose-exposed lungs exhibited an increase in noncellular thickening of the interstitium and a continued general hypercellularity at this time. In the high-dose exposure group, activities of the enzymes glutathione peroxidase, glutathione reductase, and the dehydrogenase of glucose 6-phosphate and 6-phosphogluconate were significantly elevated two- to fivefold at 2-4 d postexposure. When a correction was made for changes in lung cell number, significant increases were observed only in activities of the pentose-cycle dehydrogenases at 180-238% of controls. These increases suggested an enhanced ability of CdO-exposed lungs to generate the pentose-cycle products NADPH and ribose 5-phosphate, which would be needed for lipid and nucleic acid biosynthesis expected during the proliferative stages of epithelial repair. This study has demonstrated that the response to CdO exposure includes the induction of enzymatic activities that are related to antioxidant defense and lung repair.

Glucose metabolism in rat lung during exposure to hyperbaric O2.

Lung metabolism on exposure to hyperbaric oxygen was studied in rat lungs perfused with artificial media and ventilated with O2 at 0.2, 1, or 5 ATA. During the first 80 min of exposure to O2 at 5 ATA, glucose utilization increased 55%, lactate plus pyruvate production increased 45%, total CO2 production increased 47%, tissue ATP decreased 17%, and the ATP/ADP decreased 29% compared with 0.2 ATA O2. The increased CO2 production was due to a nearly twofold stimulation of pentose cycle activity whereas mitochondrial CO2 production did not change significantly. There were no significant differences in metabolism between lungs studied at 0.2 and 1 ATA O2. During the next hour of perfusion, there was a marked increase in mitochondrial CO2 production of control lungs but tissue ATP/ADP did not change significantly. With oxygen at 5 ATA, mitochondrial CO2 production increased only slightly, tissue ATP/ADP decreased further, and lungs demonstrated accumulation of edema fluid. The results indicate that exposure of lungs to hyperbaric oxygen results in stimulation of NADPH turnover through the pentose cycle and increased ATP generation, although the increased rate was not sufficient to maintain normal ATP/ADP.

Lung mitochondrial function following oxygen exposure and diethyl maleate-induced depletion of glutathione.

Diethyl maleate (DEM) pretreatment has previously been shown to result in a transient depletion of lung glutathione and an associated decrease of the time to the onset of rat mortality resulting from exposures to 100% oxygen in vivo. The effects of oxygen exposure on mitochondrial energy metabolism were assessed by measurements of ADP-stimulated rates of O2 utilization by lung homogenates prepared from untreated and DEM-treated rats following 4 and 24 hr of exposure to either air or 100% oxygen. Twenty-four hours of oxygen exposure of untreated rats resulted in significant decreases in lung homogenate ADP-stimulated rates of respiration supported by the substrates, pyruvate, isocitrate, and alpha-ketoglutarate. No changes were observed in succinate-supported respiration, indicating that oxygen exposure appears to adversely affect NAD-linked rather than FAD-linked pathways of mitochondrial energy metabolism. The decreased lung mitochondrial glutathione, observed 4 hr following DEM treatment, returned to normal levels following 24 hr of air and oxygen exposure. No effects of glutathione depletion were observed on ADP-stimulated rates of respiratory activity 4 hr following DEM treatment. The DEM-induced transient depletion of glutathione also did not result in any additional detrimental effects on mitochondrial respiratory activity following 24 hr of oxygen exposure in vivo. These results suggested that transient mitochondrial depletion of glutathione does not accelerate the oxygen-induced impairment of mitochondrial energy metabolism. The onset of mortality associated with DEM-pretreatment might therefore result from a failure of glutathione-dependent cytosolic protective mechanisms, rather than from an increased rate of oxygen-induced mitochondrial damage.

Glutathione redox status of control and cadmium oxide-exposed rat lungs during oxidant stress.

Activities of enzymes responsible for the maintenance of reduced glutathione (GSH) levels have been shown in a previous study to be increased in rat lungs following a 3-h exposure to cadmium oxide aerosols at 5.0 mg/m3. In this study, the ability of the lung to maintain levels of GSH during challenge with tert-butyl hydroperoxide (tBuOOH) was evaluated in isolated perfused lungs from control and cadmium oxide-exposed rats. Changes in glutathione redox status were indicated by measurements of nonprotein sulfhydryls (NPSH), total glutathione (1/2 GSH + GSSG), and glutathione disulfide (GSSG) in liquid nitrogen freeze-clamped lungs after 3-min infusions with 0-0.6 mM tBuOOH. In control and cadmium oxide-exposed lungs, levels of 1/2 GSH + GSSG remained constant over the range of 0-0.6 mM tBuOOH, indicating that no loss of glutathione from the system had occurred. In experiments with control lungs, levels of NPSH fell from 8.04 +/- 0.22 to 3.09 +/- 0.40 mumol/g dry weight when tBuOOH concentrations were increased from 0 to 0.6 mM (n = 20-23). In cadmium oxide-exposed lungs, NPSH levels also decreased proportionally to increases in GSSG. However, at concentrations of 0.075 and 0.15 mM tBuOOH, significantly smaller decreases in NPSH levels were observed in cadmium oxide-exposed lungs compared with controls. This protection against the GSH-depleting effects of tBuOOH might be explained by increased tissue levels of GSH-related enzymes.

Guinea pig lung inflammatory cell changes following acute ozone exposure.

The time course of inflammatory cell infiltration into guinea pig lungs following a single 4 h exposure to 2 ppm O3 was established by measuring the changing cell populations recovered by both bronchoalveolar lavage (BAL) and collagenase tissue digestion. Analysis of BAL-recovered albumin was used as an indicator of permeability damage and demonstrated an increase immediately following ozone exposure, reaching a maximum within 24 h, but returning to air-control levels by 7 days post-ozone exposure. A twofold enhancement in macrophages was observed in the lavage-recovered cell population after 2 days, returning to air-control numbers by 7 days. Collagenase digest-recovered monocytes and macrophages, identified by nonspecific esterase staining, were found to be elevated between 2 and 14 days following O3 exposure. Immediately following O3 exposure, a 4.5-fold increase in collagenase digest-recovered neutrophils was observed, with a subsequent decline to air-exposed lung levels during the next 12 h. In contrast, BAL-recovered neutrophils were observed to be increased immediately following O3 exposure at a level that was sustained for up to 3 days. The tissue accumulation of neutrophils was not associated with their subsequent appearance in the lavageable spaces. Although significant increases in collagenase digest-recovered eosinophils could not be detected, lavage-recovered eosinophil numbers were transiently increased by threefold after 3 days. By employing both BAL and collagenase digestion to evaluate this model of reversible lung injury, this study demonstrated that the use of BAL-recovered cell measurements alone does not adequately reflect the early inflammatory cell changes taking place within oxidant-exposed lungs.

Influenza virus infection, ozone exposure, and fibrogenesis.

Oxidant exposure following chemically induced lung injury exacerbates the tendency to develop pulmonary fibrosis. Influenza virus pneumonitis causes severe acute lung damage that, upon resolution, is followed by a persistent alveolitis and parenchymal changes characterized by patchy interstitial pneumonia and collagen deposition in the affected areas. To determine whether oxidant exposure exacerbates the virus-induced alveolitis and residual lung damage, mice were infected by aerosol inhalation with influenza A virus and continuously exposed to 0.5 ppm ozone or ambient air. Noninfected control mice were exposed to either ambient air or ozone. On various days during the first month after infection, groups of mice were sacrificed and their lungs assessed for acute injury (lung lavage albumin, total and differential cell counts, wet/dry ratios, and morphometry). At 30, 60, 90, and 120 days after infection, groups of mice were sacrificed for total and differential lavage cell counts, lung hydroxyproline content, and morphometric analysis. Ozone exposure did not alter the proliferation of virus in the lungs as quantitated by infectious virus titers of lung homogenates at 1, 4, 7, 10, and 15 days after virus infection but mitigated the virus-induced acute lung injury by approximately 50%. After Day 30 a shift in the character of the pulmonary lesions was observed in that continuous exposure to ozone potentiated the postinfluenzal alveolitis and structural changes in the lung parenchyma. Additional studies suggest that the mechanism for the enhanced postinfluenzal lung damage may be related to the oxidant impairing the repair process of the acute influenzal lung damage. These data demonstrate that ozone exposure mitigates acute virus-induced lung injury and potentiates residual lung damage.

Inflammatory cell influx into ozone-exposed guinea pig lung interstitial and airways spaces.

As a model of non-immunological lung inflammation, guinea pigs were exposed to 2.0 ppm ozone for 4 hours. Polymorphonuclear leukocytes (PMNs) rapidly accumulated in lung interstitium but declined from 147 +/- 34 million cells to control values (33 +/- 6 million cells) within the first 24 hours. Bronchoalveolar lavage (BAL) recovered PMNs were maximal by 3-6 hours (4 +/- 1 million cells) and remained elevated for 3 days. Macrophage numbers were doubled in lavageable spaces but those in interstitium increased by only a third at 2 days post-exposure. By 7 days post-exposure BAL macrophages had declined to control values of 12 +/- 2 million cells while those in interstitium remained elevated through 14 days. These data demonstrate that BAL does not necessarily reflect cellular changes in lung interstitium.

Incorporation of glucose carbons into rat lung lipids after exposure to 0.6 ppm ozone.

Continuous exposure to low concentrations of ozone has previously been associated with proliferation of lung alveolar type II epithelial cells. In this study, 14C incorporation into tissue lipids was determined in isolated rat lungs by perfusion with [U-14C]glucose, at a time of maximal hyperplasia brought about by 3 days continuous exposure to 0.6 ppm ozone. Ozone exposed lungs exhibited increased rates of glycolytic energy production, indicated by an 89% increase in 3H2O generation on perfusion with [5-3H]glucose, from a control value of 17.5 +/- 2.1 mumol X h-1 X g-1 X dry wt-1 (+/- SE, n = 4). Ozone exposure resulted in enhanced 14C incorporations into glyceride-glycerol and fatty acid moieties of lung lipids of 95 and 180%, respectively, with a greater proportion of label being recovered in shorter chain fatty acids. Although increased labeling was observed in both neutral and phospholipids, the pattern of 14C recovery suggested a relative increased glucose carbon incorporation into lung free fatty acids, phosphatidic acid, and such membrane associated lipids as phosphatidylinositol and those containing sphingosine. These results are consistent with the needs of a dividing cell population for enhanced energy production and synthesis of new lipids.

Rat lung metabolism after 3 days of continuous exposure to 0.6 ppm ozone.

Continuous exposure of rats to low concentrations of ozone has previously been associated with enhanced metabolic enzyme activities, when measured in lung homogenates. In this study, metabolic rates were measured in intact perfused lungs with altered pathology brought about by 3 days continuous exposure to 0.6 ppm ozone. Increased metabolism of ozone-exposed lungs was indicated by a twofold enhancement in glucose utilization, associated with a 62% increase in lactate formation and a 166% increase in the rate of 14CO2 production from D-[U-14C]glucose from control values of 5.2 +/- 0.5 mumol lactate and 4.4 +/- 0.6 mumol 14CO2/h per lung (+/- SE, n = 4), respectively. Mitochondrial metabolism was separately assessed by measurements of 14CO2 production from [U-14C]-pyruvate, which was found not to be significantly altered by ozone exposure, although homogenate oxygen uptake in the presence of succinate was significantly enhanced by 57%. These changes in intermediary metabolism could be correlated with increased glucose carbon incorporation into lipid and elevated activity of glucose-6-phosphate dehydrogenase. The observed elevated metabolic rates were consistent with the energy and synthetic needs of a lung during repair of ozone-induced damage.